Jurkat T cells activated by the phosphotyrosine phosphatase inhibitors H2O2 or vanadate were found to have a similar pattern of tyrosine phosphorylation when compared with T cells stimulated by anti-CD3 Ab cross-linking, suggesting that protein tyrosine phosphatase (PTP) inhibitors affect the early steps of TCR signaling. To study the role of PTPs in the most proximal membrane events of tyrosine phosphorylation, subcellular fractions of T cells were treated with the PTP inhibitors in the presence of ATP. In the membrane fraction, tyrosine phosphorylation of Lck, Fyn, and CD3 zeta can be induced by PTP inhibitors, but not by anti-CD3. Detailed characterization of this cell-free system showed that the pattern and the order of induced tyrosine phosphorylation is similar to that induced in intact cells. Upon removal of the PTP inhibitor, the tyrosine-phosphorylated proteins, including Lck, Fyn, Syk, Zap70, and CD35 zeta are rapidly dephosphorylated. Preliminary characterizations indicate that a PTP distinct from CD45, SHP1, and SHP2 is present in T cell membranes and the inhibition of this yet unidentified PTP is most likely responsible for the Lck-dependent tyrosine phosphorylation triggered by PTP inhibitors.